The present invention relates to solid substrates and to processes of making and using them in the context of separation science and analytical biochemistry.
The rapid development of and increasing need for proteins such as monoclonal and polyclonal antibodies have spawned a variety of techniques for isolating antibodies from solutions that contain them. Separation technologies such as precipitation methods, electrophoretic separations, and membrane filtration have been advanced to meet this need. The most promising technology is liquid chromatography.
Classic liquid chromatography techniques, utilizing adsorbents such as ion exchangers, hydrophobic supports, hydroxyapatite, and gel filtration media, are time-consuming and tedious to perform. Also, these techniques often lack the requisite specificity for separating heterogeneous mixtures of immunoglobulins.
Consequently, conventional techniques for separating proteins, such as immunoglobulins, have increasingly employed so-called affinity or pseudo-affinity chromatography techniques, which rely on specific interactions between an immobilized ligand and a particular molecule, such as a protein, to effect purification. Adsorbents comprising protein affinity ligands are generally effective, as described, for example, by Schwarz et al. in WO 95/31279. Yet protein-based adsorbents can present serious drawbacks. Among these are: adverse interactions with biologically active eluants; the release of protein from a solid support, which results in contamination of the desired material; chemical and physical sensitivity (e.g., to extreme pH, detergents, chaotropics, high temperature) that discourages the requisite, frequent cleaning of columns, low selectivity; and prohibitively high cost.
Known pseudoaffinity-based separation methods employ a variety of lower molecular weight ligands to effect separation. See generally U.S. Pat. Nos. 5,652,348, 5,185,313, 5,141,966, 4,701,500 and 4,381,239. These techniques typically exploit one or both of hydrophobic and electrostatic interactions, to adsorb and desorb target molecules.
Hydrophobic-interaction chromatography generally requires the addition of lyotropic salts, which presents a significant disadvantage when large-scale separations are desired. Approaches that rely on electrostatic interactions between a target molecule and an adsorbent generally require pH adjustment of the feed stock.
Lowering the pH of feed stock will ionize some ligands, such that a target molecule will adsorb. In other scenarios at low pH, other target molecules will desorb. See E. Boschetti, J. Biochem. Biophys. Methods 49 (2001) 361. But acidic pH is not always desirable, since it may induce the formation of aggregates and reduce the biological activity of some molecules such as immunoglobulins. Despite the utility and low cost of some pseudoaffinity-based ligands, therefore, most methods that employ them suffer from low capacity and the need to change feed stock ionic strength, pH, or both.
In principle, powerful analytical methods can be realized with the affinity ligand adsorbents discussed above. For example, the rapid identification of disease markers by analyte/adsorbent interactions would supplant the tedious and time consuming work required in conventional clinical diagnostics in order to prepare reagents that specifically bind to such markers. Additionally, the direct and rapid identification of differentially expressed proteins would be a significant benefit to the field, thereby circumventing, for example, the long process of polypeptide isolation and subsequent immunization to produce desired immunoglobulins. The above-mentioned shortcomings of conventional adsorbents limit the sensitivity and resolution of such analytical tools, however.
Accordingly, a need exists in the art for improved adsorbents that exhibit high binding capacity and specificity, that can be regenerated extensively without suffering physiochemical degradation, and that can function under physiological pH and/or ionic strength. A need also exists for improved biochemical analytic tools that are useful for the rapid identification of biologically important molecules.